Requirement for Host RNA-Silencing Components and the Virus-Silencing Suppressor when Second-Site Mutations Compensate for Structural Defects in the 3′ Untranslated Region

Abstract

Turnip crinkle virus (TCV) contains a structured 3= region with hairpins and pseudoknots that form a complex network of noncanonical RNA:RNA interactions supporting higher-order structure critical for translation and replication. We investigated several second-site mutations in the p38 coat protein open reading frame (ORF) that arose in response to a mutation in the asymmetric loop of a critical 3= untranslated region (UTR) hairpin that disrupts local higher-order structure. All tested second-site mutations improved accumulation of TCV in conjunction with a partial reversion of the primary mutation (TCV-rev1) but had neutral or a negative effect on wild-type (wt) TCV or TCV with the primary mutation. SHAPE (selective 2=-hydroxyl acylation analyzed by primer extension) structure probing indicated that these second-site mutations reside in an RNA domain that includes most of p38 (domain 2), and evidence for RNA:RNA interactions between domain 2 and 3=UTR-containing domain 1 was found. However, second-site mutations were not compensatory in the absence of p38, which is also the TCV silencing suppressor, or in dcl-2/dcl4 or ago1/ago2 backgrounds. One second-site mutation reduced silencing suppressor activity of p38 by altering one of two GW motifs that are required for p38 binding to double-stranded RNAs (dsRNAs) and interaction with RNA-induced silencing complex (RISC)-associated AGO1/AGO2. Another second-site mutation substantially reduced accumulation of TCVrev1 in the absence of p38 or DCL2/DCL4. We suggest that the second-site mutations in the p38 ORF exert positive effects through a similar downstream mechanism, either by enhancing accumulation of beneficial DCL-produced viral small RNAs that positively regulate the accumulation of TCV-rev1 or by affecting the susceptibility of TCV-rev1 to RISC loaded with viral small RNAs. IMPORTANCEGenomes of positive-strand RNA viruses fold into high-order RNA structures. Viruses with mutations in regions critical for translation and replication often acquire second-site mutations that exert a positive compensatory effect through reestablishment of canonical base pairing with the altered region. In this study, two distal second-site mutations that individually arose in response to a primary mutation in a critical 3= UTR hairpin in the genomic RNA of turnip crinkle virus did not directly interact with the primary mutation. Although different second-site changes had different attributes, compensation was dependent on the production of the viral p38 silencing suppressor and on the presence of silencing-required DCL and AGO proteins. Our results provide an unexpected connection between a 3= UTR primary-site mutation proposed to disrupt higher-order structure and the RNA-silencing machinery.T he structure of the genomic RNA (gRNA) of positive-strand RNA viruses participates in many basic functions, including replication, translation, and regulation of the competing processes of translation and transcription (1-3). Studies examining the secondary structure and tertiary in...

Folding behavior of a T-shaped, ribosome-binding translation enhancer implicated in a wide-spread conformational switch

“…As shown in Figures 1A,C and D , flexible residues were mainly found in TSS terminal and internal loops. Ψ 3 , which was originally identified by generating several different compensatory mutations in full-length gRNA, was not resolved by in-line probing of TSS118, similar to what was previously found when subjecting larger fragments to in-line probing and full-length gRNA to SHAPE structure probing ( Chattopadhyay et al, 2015 ; McCormack et al, 2008 ; Yuan et al, 2009 ).…”

An RNA Element That Facilitates Programmed Ribosomal Readthrough in Turnip Crinkle Virus Adopts Multiple Conformations

“…SHAPE structure probing. Structure probing was performed using SHAPE (selective 2=-hydroxyl acylation analyzed by primer extension) as described previously (42). Briefly, for in vitro SHAPE, 6 pmol of in vitrotranscribed, full-length TCV gRNA was denatured for 5 min at 65°C, snap-cooled on ice, and then incubated in folding buffer at 37°for 20 min.…”

Roles of Dicer-Like Proteins 2 and 4 in Intra- and Intercellular Antiviral Silencing

“…CP is a strong viral suppressor of RNA silencing (VSR; Chattopadhyay et al, 2015;Mérai et al, 2006;Pérez-Cañamás and Hernández, 2015;Qu et al, 2003;Thomas et al, 2003;Zhang et al, 2012). It is also required for cell-to-cell movement of TCV in N. benthamiana (Cohen et al, 2000;Li et al, 2009).…”